This invention relates to drug delivery. More particularly, this invention relates to compositions and methods or delivering drugs to tumor cells.
Cancer cells are often characterized by specific antigens or over-expressed receptors on cell surfaces. Such receptors provide uptake routes for nutrients and signals from the surrounding environment, which nutrients and signals are essential for active growth of the cells. Examples include receptors for folic acid, vitamin B12, and transferrin. S. D. Weitmann et al., Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues, 52 Cancer Res. 3396-3401 (1992); J. F. Ross et al., Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines. Physiologic and clinical implications, 73 Cancer 2432-2443 (1994); J. Holm et al., High-affinity folate binding in human choroid plexus. Characterization of radioligand binding, immunoreactivity, molecular heterogeneity and hydrophobic domain of the binding protein. 280 Biochem. J. 267-272 (1991).
Active internalization into cells of nutrients, signal molecules, or nanoparticulates via various processes, such as endocytosis and phagocytosis, is a natural consequence of evolution for survival of individual cells, signal transduction pathways, and cellular immune reactions. These processes have now been utilized as an important delivery mode for macromolecular and/or nano-sized carriers, which ferry anticancer drugs, antisense oligonucleotides, genes, or proteins, to tumor cells to kill cells and/or suppress aggressive cell growth. P. S. Low & R. J. Lee, Folate-mediated tumor cell targeting of liposome-entrapped doxorubicin in vitro, 1233 Biochem. Biophys. Acta 134-144 (1995); D. Goren et al., Nuclear delivery of doxorubicin via folate-targeted liposomes with bypass of multidrug-resistance efflux pump, 6 Clinical Cancer Res. 1949-1957 (2000).
Ligand-receptor and antigen-antibody interactions are important modes in active tumor targeting and are frequently utilized for designing carrier systems containing cytostatic agents that minimize undesirable side effects of the drugs. S. P. Vyas et al., Ligand-receptor mediated drug delivery: an emerging paradigm in cellular drug targeting, 18 Critical Review in Therapeutic Drug Carrier System 1-76 (2001).
Before ligand-receptor interactions occur, the anticancer drug carriers should (a) avoid active and massive removal processes in the body, which remove foreign particulates (carriers), and (b) minimize interactions with normal, healthy cells. One approach for accomplishing these requirements is surface decoration of the carriers with hydrophilic polymers, particularly with poly(ethylene glycol) (PEG). Such surface decoration minimizes the adsorption of plasma proteins that mediate particulate uptake (opsonization) by the reticuloendothelium system (RES). This provides a certain degree of stealth effect for particulate carriers, which provides prolonged circulation in the blood stream. Prolonged circulation in the blood stream, in turn, results in a higher probability of the drug reaching tumor sites by the enhanced permeability and retention (EPR) effect in the defective and leaky vasculature in the tumors. M. J. Roberts et al., Chemistry for peptide and protein PEGylation, 54 Adv. Drug Deliv. Rev. 459-476 (2002); G. Molineux, Pegylation: engineering improved pharmaceuticals for enhanced therapy, Cancer Treat Rev. Suppl A:13-16 (2002). The introduction of a ligand to the ends of PEG chains immobilized to the carrier surface is also employed in the design of anticancer carriers for active targeting or internalization.
Receptor-mediated endocytosis has received major attention in the field of drug/gene delivery in the past few decades. This process could be exploited for designing compositions and methods for delivery of site-specific bioactive agents, in particular, for the delivery of anti-cancer agents, because anti-cancer agents have disadvantageous properties, such as nonspecific toxicity, lack of tumor selectivity, and development of multi-drug resistance in various tumor cells. To overcome these problems and to increase the therapeutic index of the drugs, various tumor targeting moieties, such as vitamins, sugars, and antibodies, have been investigated. These target moieties showed to some extent the potential for the effective treatment of tumors. Recently, some kinds of vitamins and sugars have been used as tumor-targeting moieties, because their activities, which can internalize with macromolecules in tumor cells, were confirmed by in vitro tests. However, the successful tumor targeting of carriers with the moieties in vivo has rarely been reported. A reason for this, although tumor targeting combined with receptor-mediated endocytosis is effective, is the same receptors are present on various normal cell surfaces, with various degrees of expression. For instance, even though the receptor of folic acid, a B vitamin, is known to be over-expressed on cancer cells (e.g., ovarian, breast, lung, kidney, and brain tumors), it is also expressed on normal cells because folic acid is an essential nutrient. S. D. Garbis et al., Determination of folates in human plasma using hydrophilic interaction chromatography-tandem mass spectrometry, 73 Anal. Chem. 5358-5364 (2001). Actually, it was reported that the folate binding protein (albumin) was greatly accumulated in liver and kidney compared to controls (albumin without folate) in tumor and non-tumor bearing mouse models, while accumulation in tumor sites was slightly increased. The reason for this observation is unknown, however, two possibilities include: (i) the total amount of folate receptors expressed in liver and kidney is more than that in tumors, even though the folate receptor is over-expressed in tumors, and (ii) the likelihood of folate contacting and binding to liver and kidney is higher than that of contacting and binding to tumors. This reasoning is supported by a report that suggested that liver and kidney are the main organs for folate homeostasis. This means that folate molecules bonded to macromolecules could not serve very well as tumor-targeting moieties in vivo. In particular, in the case of a sugar moiety as a targeting ligand, this phenomenon is extreme. The receptors for sugar moieties such as glucose, galactose, and mannose are distributed at each specific organ. Thus, tumor-site targeting using a sugar moiety is very difficult, because their receptors are also expressed at normal sites. For example, even if the hepatic asialoglycoprotein receptor, found only on hepatocytes (about 500,000 receptor per cell), were a very useful receptor for liver targeting, the targeting of liver tumors using this receptor has been seldom or never reported because the receptor is distributed on all hepatic cells (tumor and normal cells). K. A. Deal et al., Cellular distribution of 111In-LDTPA galactose BSA in normal and Asialoglycoprotein receptor-deficient mouse liver, 25 Nuclear Med. & Biology 379-385 (1998). Thus, active internalization via receptor-mediated endocytosis is not specific in nature to tumor cells and inevitably causes side effects once internalized into normal cells. Therefore, to design a more effective delivery system, the development of a carrier system, which can expose a ligand to specific-site as tumor site, is necessary.
The extracellular pH (pHe) of normal tissues and blood is kept constant at pH 7.4 and pH 7.5, respectively, and their intracellular pH (pHi) is kept constant at pH 7.2. However, in most tumors the pH gradient is reversed (pHi>pHe). Particularly, pHe is more acidic in tumors than in normal tissues. I. F. Tannock & D. Rotin, Acid pH in tumors and its potential for therapeutic exploitation, 49 Cancer Res. 4373-4384 (1989); S. K. Hobbs et al., Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment, 95 Proc. Nat'l Acad. Sci. USA 4607-4612 (1998); M. Stubbs et al., Causes and consequences of tumour acidity and implications for treatment, 6 Opinion 15-19 (2000). Although there is variation in in vivo pHe measurements of human patients having various solid tumors (e.g., adenocarcinoma, squamous cell carcinoma, soft tissue sarcoma, and malignant melanoma) in readily accessible areas (limbs, neck, or chest wall) using needle type microelectrodes, the mean pH value is reported to be 7.06, with a full range of 5.66-7.78. K. Engin et al., Extracellular pH distribution in human tumors, 11 Int. J. Hyperthermia 211-216 (1995). The variation is dependent on tumor histology and tumor volume. Recent measurements of pHe by noninvasive technology, such as 19F, 31P, or 1H probes for magnetic resonance spectroscopy in human tumor xenografts in animals, further verified consistently low pHe. R. van Sluis et al., In vivo imaging of extracellular pH using 1H MRSI, 41 Magn. Reson. Med. 743-750 (1999); A. S. E. Ojugo et al., Measurement of the extracellular pH of solid tumours in mice by magnetic resonance spectroscopy: a comparison of exogenous 19F and 31P probes, 12 NMR Biomed. 495-504 (1999). All measurements of pHe of human and animal solid tumors by either invasive or noninvasive methods showed that more than 80% of all measured values falls below pH 7.2. This tumor pH can be further lowered by hyperglycemia. D. B. Leeper et al., Human tumor extracellular pH as a function of blood glucose concentration, 28 Int. J. Radiat. Oncol. Biol. Phys. 935-943 (1994).
The high rate of glycolysis of tumor cells under either aerobic or anaerobic condition has been thought to be a major cause of low pHe. The tumor cells synthesize ATP by both mitochondrial oxidative phosphorylation and glycolysis. Glycolysis produces two moles of lactic acids (pKa=3.9) and two moles of ATP by consuming one mole of glucose. Hydrolysis of ATP also produces protons. Despite the high production rate of protons in tumor cells, their cytosolic pH, particularly of resistant cells, remains alkaline, which is favorable for glycolysis, by exporting protons out of the cells by unknown mechanisms. This leads to low pHe with the help of inadequate blood supply, poor lymphatic drainage, and high interstitial pressure in tumor tissues. I. F. Tannock & D. Rotin, supra; S. K. Hobbs et al., supra; M. Stubbs et al., supra. However, it is of interest to note that glycolysis-deficient variant cells (lacking lactate dehydrogenase) produced negligible quantities of lactic acid but still presented an acidic environment. M. Yamagata et al., The contribution of lactic acid to acidification of tumours: studies of variant cells lacking lactate dehydrogenase, 77 Br. J. Cancer 1726-1731 (1998). This finding has been interpreted as meaning that acidity is a phenotype of tumor cells rather than a consequence of cell metabolic events. The acidic environment may benefit the cancer cells, because it promotes invasiveness (metastasis) by destroying the extracellular matrix of surrounding normal tissues.
This tumor pHe can be further manipulated to shift to even lower pH by administration of glucose. The rate of glucose uptake by tumor cells is higher than that of normal cell due to higher metabolism and greater production and secretion of lactic acid. This can lead to lower pH of the extracellular matrix and can be beneficial for the approaches described in this application.
In view of the foregoing, it will be appreciated that providing a tumor-selective targeting system for drug delivery would be a significant advancement in the art.